Genetic analysis of erythrocyte superoxide dismutase polymorphism in the genus Cervus

A study of erythrocyte superoxide dismutase in Cervus dama, Cervus elaphus, Cervus nippon and Cervus elaphus x Cervus nippon hybrids has revealed a polymorphism of this enzyme system in Cervus elaphus as well as in the hybrid population. Genetic analysis suggests that this enzyme system is controlled by one gene locus with two codominant alleles. The allele frequencies allow a clear discrimination between the hybrid population and the red deer population, whereas the fallow deer are fixed for the allele which is most common in red deer. The comparison of the genotypic structures with the Hardy-Weinberg structure shows a slight excess of homozygotes in all populations, but the deviation is significant only for the hybrid population as well as one red deer population. Genetic variation within and differentiation between demes is quantified using different measures. Finally, some management implications of these first results are discussed.     

Phylogenetic relationships among european red deer, wapiti, and sika deer inferred from mitochondrial DNA sequences

We determined the mitochondrial cytochrome b gene sequences (1140 bp) of one subspecies of the European red deer (Cervus elaphus in Europe), three subspecies of the wapiti (C. elaphus in Asia and North America), and six subspecies of the sika deer (C. nippon in Japan). Our phylogenetic analysis revealed the monophyly of the European red deer, that of the wapiti, and that of the sika deer. The wapiti, however, was shown to be more closely related to the sika deer than to the European red deer. This is in conflict with traditional morphological results, which suggest a close sister group relationship between the wapiti and the European red deer. The divergence time between the European red deer and the wapiti plus the sika deer was estimated to be approximately 0.80 Ma, and that between the wapiti and the sika deer was estimated to be 0.57 Ma. The sika deer was subdivided into two subspecies groups, and the wapiti was also found to consist of an Asian group and a North American group.     

A phylogenetic comparison of red deer and wapiti using mitochondrial DNA

A phylogeny was constructed for red deer/wapiti (Cervus elaphus) subspecies using sequence data from the control region of mitochondrial DNA (mtDNA). The tree was rooted using Cervus nippon (sika deer), Cervus albirostris (Thorold's white-lipped deer), and several Odocoileinae species. A division between the mtDNA haplotypes of red deer (European) and wapiti (Asian/North American) corresponds to subspecies found on opposite sides of the Himalayan Mountains and Gobi, which suggests wapiti should be reconsidered for the status of C. canadensis. Using parsimony and distance analysis, red deer and wapiti are derived from a single recent common ancestor, which is consistent with current taxonomy that recognizes the subspecies of Cervus elaphus as monophyletic group. However, maximum-likelihood analysis using weighted transitional substitutions caused red deer to form a sister group to sika deer (Cervus nippon) and wapiti. A phenetic comparison revealed wapiti also share more nucleotide similarities with sika deer, although approximately 5% sequence divergence separates wapiti, sika, and red deer. Phylogenetic evidence from the cytochrome b sequences corroborated observations from the control region. Observations from this study suggest that the species status of wapiti should be reinstated.     

Introgression through rare hybridization: A genetic study of a hybrid zone between red and sika deer (genus Cervus) in Argyll, Scotland

In this article we describe the structure of a hybrid zone in Argyll, Scotland, between native red deer (Cervus elaphus) and introduced Japanese sika deer (Cervus nippon), on the basis of a genetic analysis using 11 microsatellite markers and mitochondrial DNA. In contrast to the findings of a previous study of the same population, we conclude that the deer fall into two distinct genetic classes, corresponding to either a sika-like or red-like phenotype. Introgression is rare at any one locus, but where the taxa overlap up to 40% of deer carry apparently introgressed alleles. While most putative hybrids are heterozygous at only one locus, there are rare multiple heterozygotes, reflecting significant linkage disequilibrium within both sika- and red-like populations. The rate of backcrossing into the sika population is estimated as H = 0.002 per generation and into red, H = 0.001 per generation. On the basis of historical evidence that red deer entered Kintyre only recently, a diffusion model evaluated by maximum likelihood shows that sika have increased at approximately 9.2% yr-1 from low frequency and disperse at a rate of approximately 3.7 km yr-1. Introgression into the red-like population is greater in the south, while introgression into sika varies little along the transect. For both sika- and red-like populations, the degree of introgression is 30-40% of that predicted from the rates of current hybridization inferred from linkage disequilibria; however, in neither case is this statistically significant evidence for selection against introgression.     

Introgression of exotic Cervus (nippon and canadensis) into red deer (Cervus elaphus) populations in Scotland and the English Lake District

Since the mid‐19th century, multiple introductions of Japanese sika deer (Cervus nippon nippon) and North American wapiti (C. canadensis) have taken place in the British Isles. While wapiti have generally been unsuccessful, sika have been very successful, especially in Scotland where they now overlap at least 40% of the range of native red deer (C. elaphus). Hybridization between these two species and red deer has been demonstrated in captivity and in the wild. Using a panel of 22 microsatellite loci that are highly diagnostic between red deer and sika, and moderately diagnostic between red deer and wapiti, we investigated the extent of introgression between these species in 2,943 deer sampled from around Scotland and from the English Lake District using the Bayesian clustering software STRUCTURE. We also used a diagnostic mitochondrial marker for red deer and sika. Our survey extends previous studies indicating little introgression of wapiti nuclear alleles into red deer, in particular in Northern Scotland, Kintyre, and the Lake District. We found a new area of extensive sika introgression in South Kintyre. In the North Highlands, we show for the first time geographically scattered evidence of past hybridization followed by extensive backcrossing, including one red‐like individual with sika introgression, two sika‐like individuals with red deer introgression, and six individuals that were apparently pure sika at the nuclear markers assessed but which carried red deer mitochondria. However, there has not been a collapse of assortative mating in this region. Similarly, in the English Lake District red deer, we found only traces of past sika introgression. No sika alleles were detected in the Central Highlands or the Hebridean red deer refugia. We make suggestions for management to prevent further spread of sika alleles into red deer and vice versa.     

A survey of the hybridisation status of Cervus deer species on the island of Ireland

Red deer (Cervus elaphus) did not recolonise Ireland after the last glaciation, but the population in Co. Kerry is descended from an ancient (c. 5000 BP) introduction and merits conservation. During the mid-19th century exotic species including North American wapiti (C. canadensis) and Japanese sika deer (C. nippon nippon) were introduced to Ireland, mainly via Powerscourt Park, Co. Wicklow. While wapiti failed to establish, sika thrived, dispersed within Co. Wicklow and were translocated to other sites throughout Ireland. Red deer and sika are known to have hybridised in Ireland, particularly in Co. Wicklow, but an extensive survey with a large, highly diagnostic marker panel is required to assess the threat hybridisation potentially poses to the Co. Kerry red deer population. Here, 374 individuals were genotyped at a panel of 22 microsatellites and at a single mtDNA marker that are highly diagnostic for red deer and Japanese sika. The microsatellites are also moderately diagnostic for red deer and wapiti. Wapiti introgression was very low [trace evidence in 2 (0.53 %) individuals]. Despite long-standing sympatry of red deer and sika in the area, no red deer-sika hybrids were detected in Co. Kerry suggesting strong assortative mating by both species in this area. However, 80/197 (41 %) of deer sampled in Co. Wicklow and 7/15 (47 %) of deer sampled in Co. Cork were red-sika hybrids. Given their proximity and that hybrids are less likely to mate assortatively than pure individuals, the Co. Cork hybrids pose a threat to the Co. Kerry red deer.     

A genetic study of sika (Cervus nippon) in the New Forest and in the Purbeck region, southern England: is there evidence of recent or past hybridization with red deer (Cervus elaphus)?

Sika Cervus nippon are native to Japan and East Asia but are now naturalized in many parks of the world, including Britain. In contrast to the substantial body of research on the extent of hybridization between sika and red deer Cervus elaphus in Scottish populations, there has been little genetic analysis of the English populations of sika. Sika in England still have a patchy and discontinuous distribution; populations are thus still genetically isolated and may be expected to show higher variability in genetic type. The current paper uses DNA-based techniques to explore the genetic composition of sika in the New Forest (Hampshire) and that of sika from the largest population in England, in the Purbeck region (Dorset). The study aims were to determine whether New Forest sika show signs of recent interbreeding with New Forest red deer populations, whether New Forest sika are genetically distinct from Purbeck sika, and whether New Forest and Purbeck sika show evidence of past hybridization or whether they are pure (non-hybrid) strains. Microsatellite analysis was used to compare the genetic profiles of individual deer. Results showed that sika and red deer in the New Forest were genetically distinct, indicating that there is no large-scale in situ hybridization occurring between these feral populations. In terms of overall genetic composition, there was no significant difference between the sika in the New Forest and Purbeck. However, a more detailed analysis found that New Forest sika showed a lower level of introgression with red deer compared with the Purbeck sika. We conclude that, overall, the New Forest sika deer do appear to be more genetically pure bred than the Purbeck sika.     

白唇鹿的染色体组型、C带和G带带型

白唇鹿产于我国西藏东部和四川西部。它和大熊猫一样,是我国特有的珍贵动物。有关它的染色体组型至今尚未见报道。我们于去年八月,对两头雄性白唇鹿外周血及茸缸淋巴细胞进行了培养,并作了染色体组型观察和C带及G带带型的观察。 一、材料和方法 北京动物园的两头准性白唇廘,一头10岁,一头4岁。 每一头先后采样二次。用装有司可林(Succinylcholinum chloratum)麻醉弹的麻醉枪击中鹿后,在颈静脉处,用含有1毫升肝素(浓度为50微     

The establishment of a hybrid zone between red and sika deer (genus Cervus)

Japanese sika deer ( Cervus nippon nippon ) were introduced to Scotland around 80 years (20 generations) ago. The sika phenotype is expanding its range and hybridizing extensively with native red deer ( Cervus elaphus ) leading to the establishment of a hybrid zone. This zone is currently moving and cannot be considered to be at equilibrium. Cervid genotypes and mitochondrial haplotypes were mapped across the sika phenotype range, using diagnostic protein isozymes, microsatellite nuclear DNA markers and RFLPs in mtDNA. These were analysed to estimate heterozygote deficits and nuclear linkage disequilibria and cytonuclear disequilibria in relation to gene frequencies and time since contact. Introgression was found in both taxa and strong linkage disequilibria and heterozygote deficits characterize the populations longest exposed to hybridization. Populations further from the introduction site, where hybridization is facilitated by the dispersal of sika-like stags, show low values for linkage disequilibria and heterozygote deficit. The observed patterns in genotype are explained in terms of assortative mating and a selective advantage of the sika genotype. The genetic integrity of the Scottish mainland red deer is shown to be at risk from the invasion of sika.     

Variable extent of hybridization between invasive sika (Cervus nippon) and native red deer (C. elaphus) in a small geographical area

Instances of hybridization between endemic and alien species pose a threat to species integrity but also provide us with an opportunity to study the dynamics of gene flow between two species as they first meet. Here, we used variation at 22 highly differentiated microsatellite loci and one mitochondrial DNA (mtDNA) marker in a sample of 735 individuals, to investigate the genetic consequences of an introduction of Japanese sika deer ( Cervus nippon ) for native red deer ( C. elaphus ) on the Kintyre Peninsula in Scotland. We investigated population structure, estimated null-allele frequency and assigned individual hybrid scores using a Bayesian clustering algorithm implemented in structure 2.2. The dataset clearly divided into two clusters and generally, introgression into red and sika was low. However at one site, West Loch Awe, 43% of individuals were hybrids. MtDNA introgression indicated that hybridization was occurring between red-deer hinds and sika-deer stags. We argue that the pattern of differential introgression across the study area is primarily due to the rarity of hybridization events between the two species and the limited time the two species have been in contact (< 120 years). This contrasts with the causes of classic mosaic hybrid zones (selection induced by habitat variability). Currently, it seems possible that, in time, the level of hybridization found at West Loch Awe could also be found across the whole of the peninsula.     

东北马鹿和东北梅花鹿F_1杂种精母细胞联会复合体分析

作者以界面铺张-硝酸银染色技术,对东北马鹿和东北梅花鹿的F_1可育杂种的精母细胞联会复合体进行亚显微观察及分析。在减数分裂前期,杂种鹿精母细胞中形成31条完整的常染色体联会复合体、一个端着丝粒染色体/中着丝粒染色体的三价体和XY双价体。这进一步证明,两种亲本鹿的染色体具有高度的同源性,其差别仅在于一个罗伯逊易位。三价体的顺式构型可能和杂种鹿的可育性有关。     

Genetic structure of,and hybridisation between,red (Cervus elaphus) and sika (Cervus nippon) deer in Ireland

This study investigated the levels of genetic diversity and variation exhibited by red and sika deer in Ireland, along with the extent and regional location of hybridisation between these two species. Bi-parental (microsatellites) and maternally-inherited (mitochondrial DNA) genetic markers were utilised that allowed comparisons between 85 red deer from six localities and 47 sika deer from 3 localities in Ireland. Population genetic structure was assessed using Bayesian analysis, indicating the existence of two genetic clusters in sika deer and three clusters in red deer. Levels of genetic diversity were low in both red and sika deer. These genetic data presented herein indicate a recent introduction of sika deer and subsequent translocations in agreement with historical data. The origins of the current red deer populations found in Ireland, based on genetic data presented in this study, still remain obscure. All hybrid deer (red/sika) found in this study were found in Wicklow, Galway and Mayo where the ‘red-like’ deer exhibited sika deer alleles/haplotypes, and vice versa in the case of Wicklow. Molecular methods proved invaluable in the identification of the hybrid deer because identification of hybrids based on phenotypic external appearances (pelage and body proportions) can be misleading. Areas where red and sika deer are sympatric need to be assessed for the level and extent of hybridisation occurring and thus need to be managed in order to protect the genetic integrity of ‘pure’ red deer populations.     

Variability of Female Responses to Conspecific vs. Heterospecific Male Mating Calls in Polygynous Deer: An Open Door to Hybridization?

Males of all polygynous deer species (Cervinae) give conspicuous calls during the reproductive season. The extreme interspecific diversity that characterizes these vocalizations suggests that they play a strong role in species discrimination. However, interbreeding between several species of Cervinae indicates permeable interspecific reproductive barriers. This study examines the contribution of vocal behavior to female species discrimination and mating preferences in two closely related polygynous deer species known to hybridize in the wild after introductions. Specifically, we investigate the reaction of estrous female red deer (Cervus elaphus) to playbacks of red deer vs. sika deer (Cervus nippon) male mating calls, with the prediction that females will prefer conspecific calls. While on average female red deer preferred male red deer roars, two out of twenty females spent more time in close proximity to the speaker broadcasting male sika deer moans. We suggest that this absence of strict vocal preference for species-specific mating calls may contribute to the permeability of pre-zygotic reproductive barriers observed between these species. Our results also highlight the importance of examining inter-individual variation when studying the role of female preferences in species discrimination and intraspecific mate selection.     

Affinities between Cutifilaria (Nematoda: Filarioidea), parasites of deer, and Mansonella as seen in a new onchocercid, M. (C.) perforata n. sp., from Japan

A new dermal filarioid nematode, collected from Cervus nippon nippon (sika deer) on Kyushu Island, Japan, showed close affinities between the genera Cutifilaria and Mansonella (Onchocercidae: Onchocercinae): no buccal capsule, esophagus reduced to a thin fibrous tube, and female tail with four lappets. We propose Cutifilaria as a subgenus of Mansonella. Cutifilaria was distinguished from the five other subgenera, Mansonella, Tetrapetalonema, Esslingeria, Sandnema, and Tupainema, in having an area rugosa composed of transverse bands with tiny points, 14-16 papillae around the cloacal aperture, two prominent rhomboidal subterminal papillae, and a thick right spicule with spoon-shaped distal extremity. The host range of Mansonella was extended to ungulates by the addition of Cutifilaria, which appears to be derived from Tupainema, parasitic in Tupaioidea (insectivores), because of the similarity in their right spicules; Cutifilaria seems to have an Asiatic origin. M. (C.) perforata n. sp. was distinct from the sole other related species, M. (C.) wenki, a parasite of Cervus elaphus (red deer) in Europe, having a more complex right spicule with a sturdy terminal point and microfilariae with a bifid posterior end. In addition, almost all females had cuticular pores near the vulva, on the ventral line. The prevalence of microfiloriae and adults of M. (C.) perforata in the skin of sika deer was 38% and 21%, respectively.     

A mitochondrial control region and cytochrome b phylogeny of sika deer (Cervus nippon) and report of tandem repeats in the control region

Sika deer (Cervus nippon Temminck) are endemic to mainland and insular Asia. Numerous subspecies have been named, but they are not quantitatively well defined. Portions of the mitochondrial cytochrome b gene (450 bp) and control region (512 bp) were sequenced from 28 individuals belonging to five sika subspecies and two Cervus elaphus subspecies. Phylogenetic trees constructed using these sequences clearly demonstrated that sika are monophyletic with respect to C. elaphus. A survey of variation in the control region showed that approximately half the variation occurred in a 100-base segment between positions 150 and 250 in the left domain of the control region. Within this region there were three tandemly repeated copies of a 39-base motif. In addition, two of the samples (C. n. aplodontus and C. n. hortulorum) contained, respectively, two and four additional copies of the repeated motif.     

Investigating temporal changes in hybridization and introgression in a predominantly bimodal hybridizing population of invasive sika (Cervus nippon) and native red deer (C. elaphus) on the Kintyre Peninsula,Scotland

We investigated temporal changes in hybridization and introgression between native red deer (Cervus elaphus) and invasive Japanese sika (Cervus nippon) on the Kintyre Peninsula, Scotland, over 15 years, through analysis of 1513 samples of deer at 20 microsatellite loci and a mtDNA marker. We found no evidence that either the proportion of recent hybrids, or the levels of introgression had changed over the study period. Nevertheless, in one population where the two species have been in contact since ～1970, 44% of individuals sampled during the study were hybrids. This suggests that hybridization between these species can proceed fairly rapidly. By analysing the number of alleles that have introgressed from polymorphic red deer into the genetically homogenous sika population, we reconstructed the haplotypes of red deer alleles introduced by backcrossing. Five separate hybridization events could account for all the recently hybridized sika‐like individuals found across a large section of the Peninsula. Although we demonstrate that low rates of F1 hybridization can lead to substantial introgression, the progress of hybridization and introgression appears to be unpredictable over the short timescales.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in the Czech Republic

In the Czech Republic, sera from 720 wild ruminants were examined for antibodies to Neospora caninum by screening competitive-inhibition enzyme-linked immunosorbent assay and confirmed by indirect fluorescence antibody test (IFAT); the same sera were also examined for antibodies to Toxoplasma gondii by IFAT. Neospora caninum antibodies were found in 14% (11 positive/79 tested) roe deer (Capreolus capreolus), 14% (2/14) sika deer (Cervus nippon), 6% (24/ 377) red deer (Cervus elaphus), 1% (2/143) fallow deer (Dama dama), 3% (3/105) mouflon (Ovis musimon), and none of 2 reindeer (Rangifer tarandus). Toxoplasma gondii antibodies were found in 50% (7/14) sika deer, 45% (169/377) red deer, 24% (19/79) roe deer, 17% (24/143) fallow deer, 9% (9/105) mouflon, and 1 of 2 reindeer. In 42 samples of wild ruminants that tested positive for N. caninum antibodies, 28 (67% of the positive N. caninum samples) reacted solely to N. caninum. This is the first evidence of N. caninum infection in mouflon, the first N. caninum seroprevalence study in farmed red deer, and the first survey of N. caninum in wild ruminants from the Czech Republic.     

Assessment of in vitro fertility of deer spermatozoa by heterologous IVF with zona-free bovine oocytes

We have previously shown that the in vitro development of deer embryos differed according to the IVF conditions. The aim of the study was to use heterologous IVF with zona-free matured bovine oocytes to assess the in vitro fertility of 3 samples of deer semen (2 ejaculates from sika deer (Cervus nippon) and 1 pool of epididymal spermatozoa from red deer (Cervus elaphus)). The frozen/thawed semen samples were selected on Percoll gradient and resuspended in Tyrode modified medium supplemented with estrus sheep serum (0, 2, 20% v/v) or heparin (10 microg/mL). During 8 h of culture, the sperm motility index according to the post-insemination time (hpi) did not differ either between samples or between supplemented IVF media. In vitro matured zona-free bovine oocytes were inseminated in different IVF media with the semen samples. Penetration rates assessed at 15 hpi were optimal with 20% estrus sheep serum for sika deer ejaculates whereas 2% were sufficient to reach the maximum functionality of epididymal spermatozoa from red deer. The mean time of pronuclear formation was similar regardless of the semen sample. The precocity of the onset of the first S-phase in both pronuclei was characterized by Bromo-deoxy-Uridine exposures between 5 and 15 hpi in order to assess the developmental potential conferred by the semen sample (intrinsic value). As we previously observed in homologous IVF, this value seemed to be higher for the epididymal sperm sample.     

Genetic differentiation between introduced Central European sika and source populations in Japan: effects of isolation and demographic events

Japanese sika deer (Cervus nippon nippon) were introduced at the turn of nineteenth and twentieth century to many countries in Eurasia, North America and Australasia. Subsequently, free-living invasive populations have become established in several countries, including the Czech Republic, where the expanding sika population causes serious problems through overgrazing, damage through browsing and through competition and hybridisation with native red deer. 122 Japanese and 221 Czech samples were used to examine the genetic diversity, genetic structure, and the level of genetic differentiation between native populations and those introduced to the Czech Republic. Analyses of 22 microsatellite loci revealed, for both countries, evidence of isolation by distance and clear sub-structuring of populations, different from patterns previously revealed by mtDNA markers. The high number of private alleles (58 within the Czech Republic and 84 within Japan), the Fst values, factorial correspondence analysis and Bayesian clustering support a high level of divergence between the source and introduced populations. Genetic variability was generally low due to recent demographic events (founder effect in the Czech population, bottlenecks in Japanese populations); however, the values of expected heterozygosity differed greatly between subpopulations and were not the lowest in the introduced Czech populations. Multiple introductions, rapid population growth, and possible hybridisation with red deer seem to have helped the successful expansion of sika within the Czech Republic. The results also indicate that male-mediated gene flow and human-mediated translocations have significantly influenced the current genetic structure of native sika populations in Japan.     

Bovine microsatellite loci are highly conserved in red deer (Cervus elaphus), sika deer (Cervus nippon) and Soay sheep (Ovis aries)

We tested 174 bovine microsatellite primer pairs for use in a primitive breed of sheep and two species of deer. Of 173 markers, 127 (73·4%) gave a product in Soay sheep ( Ovis aries ) of which 54 (42·5%) were polymorphic. One hundred and twenty-nine of 174 (74·1%) markers gave a product in red deer ( Cervus elaphus ) of which 72 (55·8%) were polymorphic. In sika deer ( Cervus nippon ) 126 of 171 (73·7%) microsatellite primers gave a product with 47 (37·3%) polymorphic. The proportion of bovine microsatellite loci conserved across artiodactyl species was significantly greater in this study than previously reported. Reasons for this high degree of microsatellite conservation are discussed. We suggest that a high resolution comparative map of the artiodactyls can be constructed using microsatellites.